Yeasts are important producing strains in alcohol and food industries. As the energy crisis and environmental awareness of the human being are increasingly enhanced, the use of fuel alcohol instead of gasoline is becoming a development trend. Heat-resistant yeasts can reduce the trouble and cost caused by cooling in alcohol production, thereby ensuring the normal proceeding of industrial fermentation at high temperature; and therefore, screening about heat-resistant yeasts becomes a hot spot of research. In recent years, many artificially bred heat-resistant yeast strains have been put into production, and have gained good economic benefits.
As early as the middle of the 20th century, Chinese microbiologists started to work on domesticated breeding and research on heat-resistant yeasts capable of being fermented by using molasses as a raw material. Currently, many artificially bred heat-resistant yeast strains have been put into production, and have gained good economic benefits. It is reported that the strains for production are domesticated and screened to obtain the heat-resistant alcohol yeast applicable to corn mash fermentation. For quite a long time, breeding of heat-resistant strains mainly depends on screening from nature and high-temperature domestication. The researchers enhance the maximum growth temperature of Saccharomyces cerevisiae by 3° C. through chemical mutagenesis and genetic recombination; by combining high-temperature domestication/ultraviolet mutagenesis and thermal shock, the fermentation temperature of the alcohol-producing yeast is increased by 6° C.; a protoplast fusion technique is utilized, so that the alcohol yield of Saccharomyces cerevisiae and respiratory-deficient heat-resistant Kluyveromyces under the condition of 45° C. can reach 7.4%; by means of high-temperature domestication, the Saccharomyces cerevisiae can grow at 40° C.; by using screening from nature and ultraviolet mutagenesis, the alcohol yield of the Saccharomyces cerevisiae under the condition of 42° C. is increased; and a normal-pressure room-temperature plasma mutagenesis technique is utilized to obtain a high-yield oleaginous yeast.
Pichia pastoris (P. pastoris for short) is a widely used eukaryon expression system, which has the obvious advantages of high exogenous protein expression level, simple nutrients, easy induction and the like, but has the defect of high energy consumption caused by low induction temperature and has the problem that the expression of exogenous proteins is obviously inhibited when the fermentation is performed under high-temperature conditions. Up to now, there is no research report that the fermentation of P. pastoris at high temperature does not influence the expression of exogenous proteins.
The optimal temperature for growth of P. pastoris is 28-30° C.; besides, previous research has shown that the reduction of induction temperature can promote the expression of exogenous proteins; and under low-temperature conditions, the activity and transcription level of alcohol oxidase AOX can be enhanced, and the apoptosis of the strain is lowered. [Sue Macauley-Patrick, Mariana L. Fazenda, Brian McNeil and Linda M. Harvey, Heterologous protein production using the Pichia pastoris expression system. Yeast 2005, 22: 249-270; Min Zhaosheng, Guo Huiming, Yan Xu, Hong Housheng. P. pastoris High-density Fermentation Research Progress, Biotechnology Bulletin, 2014, 3:42-49]. The research of Li, et al [Li Z, Xiong F, Lin Q, et al. 2001. Low temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris. Protein Expression Purification, 21(3): 438-445.] finds that after the induction temperature is lowered from 30° C. to 23° C., the expression level of the herring antifreeze protein is increased from 5.3 mg/L to 18.0 mg/L, and the cell activity is obviously enhanced. The preliminary research of the research group compares the effects of different induction temperatures in a 7 L fermentation tank on the expression level of Rhizopus chinensis leader peptide lipase in P. pastoris. The experiments indicate that under the condition of 28° C., the maximum enzyme activity can reach 1412.5 U/mL, which is as 2.3 times as that under the condition of 30° C. and as 1.3 times as that under the condition of 25° C.; it is detected that as the induction temperature becomes lower, the death rate of the strain gets lower; however, under the condition of 25° C., the growth rate of the strain is lower, and the expression of enzyme activity is affected [Wang Tongchun, Yu Xiaowei, Xu Yan, Research of Regulation of Expression and Stability of Rhizopus chinensis Leader Peptide Lipase by Induction Temperature, Industrial Microbiology, 2010, 4:34-39]. All the existing research reports show that the induction temperature for expressing exogenous proteins by using P. pastoris is controlled at 30° C. or below so as to obtain a higher exogenous protein expression level. However, the lower induction temperature requires abundant energy consumption for lowering the temperature in industrial production; and therefore, it is urgent to develop a Pichia pastoris expression strain capable of efficiently inducing the expression of exogenous proteins under high-temperature conditions.